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2 protocols using rabbit anti mouse phospho stat1

1

Multiparametric Analysis of Signaling Pathways

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Rabbit anti phospho-Smad2 (Millipore, Billerica, MA, USA), rabbit anti Smad2/3 (Cell Signaling Technology, Danvers, MA, USA), rabbit anti phospho-Smad3 (Cell Signaling Technology), goat anti phospho-Smad1/5/8 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti Smad1/5/8 (Santa Cruz Biotechnology), rabbit anti-mouse phospho-Stat1 (Cell Signaling Technology), rabbit anti-mouse ApoA-I (Santa Cruz Biotechnology), rabbit anti-mouse Actin (Sigma, Steinheim, Germany) and HRP conjugate donkey anti-rabbit IgG (Southern Biotech, Birmingham, AL, USA) were used for western blot analysis. Rabbit anti SRB1 monoclonal antibody (Novus Biologicals, CO, USA) and FITC conjugated anti-rabbit IgG (Sigma, Steinheim, Germany) were used for flow cytometry analysis. Fc block and fluorochrome conjugated antibodies against mouse CD45, CD3, CD8, MHC-II, CD11c and TNF-alpha were purchased from BD Biosciences. Intracellular staining for TNF-alpha was performed following manufacturer's instructions. For flow cytometry experiments acquisition was done using either FACSCalibur or FACSCanto II (BD Biosciences). FlowJo Software (TreeStar) was used to analyze cellular events.
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2

Immunohistochemical Analysis of Mouse Paw

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Frozen-sections of paws were fixed in 4% PFA, blocked in 8% BSA/PBS and incubated with the following primary antibodies: rabbit anti-mouse phospho-STAT1 (1:200, Cat # 9171, Cell Signaling Technology), IKK-alpha (1:200, Cat # 2682, Cell Signaling Technology), phospho-p65 (1:200, phosphorylation site S536, Cat # 3033, Cell Signaling Technology), phospho-mTOR (1:200; Cat# 5536, Cell Signaling Technology), phospho-S6 (1:200; Cat #4858, Cell Signaling Technology), LC3 (1:200, Cat # L7543, Sigma-Aldrich), phospho-ULK1 (1:200, Cat # 5869, Cell Signaling Technology), rat anti-mouse LAMP-1 (1:100, Cat # SC19992, Santa Cruz), mouse anti-mouse p62 (IgM, 1:500, Cat # MABC32, clone 11C9.2, Millipore), biotin-conjugated anti-mouse Mac-3 monoclonal antibody (1:200, Cat # CL8943AP, Cedarlane Laboratories), mouse anti-mouse ubiquitin (IgG, 1:500, Cat # 64301, clone P4D1, Biolegend) for 1 h at room temperature followed by the appropriate secondary antibodies: rhodamine red-conjugated secondary antibody (1:100, Jackson ImmunoResearch Laboratories), FITC conjugated-secondary antibody (1:100, Jackson ImmunoResearch Laboratories) or FITC conjugated-streptavidin (1:100, Cat: 7100-02, Southern Biotechnology Associates). All images were visualized on a Nikon Eclipse microscope and acquired with QCapture software.
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